Aging is associated with increased incidence of respiratory
disorders, and elderly patients represent a disproportionate number of
afflicted individuals with pneumonia, acute lung injury, and lung
fibrosis, among other lung disorders [1-5]. This association has also
been noted in experimental models of lung disease. For example,
senescent rodent lungs are more susceptible to lung injury in the
setting of mechanical ventilation, ozone exposure, and pulmonary
infection [6-10]. Furthermore, intratracheal instillation of
lipopolysaccharide results in the exaggerated expression of
proinflammatory cytokines in senescent animals when compared to young
controls [9, 11]. Senescent lungs are also more susceptible to
bleomycin-induced lung injury [12, 13]. Together, these studies point to
the enhanced susceptibility of the senescent lung to injury, but little
is known about the factors responsible for this susceptibility.

Several mechanisms have been proposed to explain the abovementioned
observations including increased oxidative stress and free radical
damage, a decline in immune responses, and alterations in stem
cell/progenitor cell differentiation potential [14-16]. Mitochondrial
dysfunction has also been implicated in aging since the coordination
between nuclear and mitochondrial communication during aging appears to
be affected [17]. There is also much literature showing alterations in
lung structure and function in the aging lung [18-21]. Consistent with
this, we previously reported that aging murine lungs harvested from old
mice are characterized by increased expression of fibronectin and
collagen matrix mRNAs and by induction of the profibrotic factor,
transforming growth factor [beta] [22]. Thus, active matrix remodeling
may account for the subtle changes observed in lung structure as well as
the decline in lung function observed in the elderly [23] and might
render the host susceptible to disrepair after lung injury [22].

Another abnormality associated with aging is a shift in the redox
states of plasma thiol-disulfide redox couples [24, 25]. Cysteine (Cys)
and its disulfide cystine (CySS) constitute the major small molecular
weight thiol-disulfide redox couple in extracellular compartments. The
redox state of the Cys/CySS couple, expressed as [E.sub.h]CySS
calculated from the Nernst equation, is about -80 mV in the plasma of
healthy young adults [26] but is more oxidized in older individuals
[24]. The mechanisms that regulate [E.sub.h]CySS and the factors that
lead to its oxidation with aging are largely unknown.

Accumulating evidence suggests that cells actively participate in
the control of their extracellular redox environment. When mammalian
cells are grown in culture, some of the CySS that is provided by the
medium is converted to Cys until the optimal redox state of -80 mV is
reached [2729]. If one replaces the CySS in the medium with Cys, the
cells will again adjust the concentrations of extracellular Cys and CySS
to achieve a redox state of -80 mV. These observations suggest that
there is an optimal set point for the extracellular redox state and that
cells have the capacity to restore this equilibrium when challenged with
either reducing or oxidizing conditions.

Fibroblasts play a key role in maintaining the extracellular
matrix, but it is unknown if they contribute to the maintenance of the
extracellular redox environment as well. We previously reported that
primary lung fibroblasts cultured in media with oxidized [E.sub.h]CySS
showed increased expression of fibronectin and TGF[beta]1, among other
changes, indicating that oxidation of the Eh CySS alters matrix gene
expression in lung fibroblasts [30]. The purpose of the present study
was to determine whether fibroblasts from old and young mice have
different extracellular [E.sub.h]CySS set points and whether these
differences translate into altered expression of extracellular
matrix-related genes.

2.2. Animals and Primary Lung Fibroblasts. Mice were housed in a
pathogen-free barrier facility accredited by the Association for
Assessment and Accreditation of Laboratory Animal Care, and procedures
were approved by the University of Louisville's Institutional
Animal Care and Use Committee. The animals used in this study were young
(2 months old) or old (24 months old) female C57BL/6 mice purchased from
Charles River Laboratories (Wilmington, MA). Primary lung fibroblasts
were harvested from these animals and cultured in DMEM supplemented with
10% FBS and 1% antibiotic-antimycotic solution (Cellgro, Manassas, VA)
as previously described [31, 32]. All experiments described here used
cells between passages numbers 3 and 8.

2.4. Culturing Cells in Redox Media. Primary lung fibroblasts from
young mice and old mice were plated in 6-well plates at a density of 8 x
[10.sup.5] cells/well in DMEM supplemented with 10% FBS and 1%
antibiotic-antimycotic solution. After 24 hours, the media on all plates
were replaced with serum-free media containing 4 mM L-glutamine, 1%
antibiotic-antimycotic solution, and Cys and CySS at concentrations that
produced one of 3 different [E.sub.h]CySS: oxidizing (0 mV), typical
(-80 mV), or reducing (-150 mV). Aliquots of each media were added to an
empty 6-well plate (for 0 hours no-cell controls) or to plates
containing young and old cells (for 0 hours time points) and immediately
removed and processed for Cys/CySS and GSH/GSSG analysis. Later time
points were collected after incubation of plates containing young, old,
or no cells for the indicated times.

2.5. Sample Collection and Analysis of Cys, CySS, GSH, and GSSG.
Conditioned media were centrifuged at 500 x g to pellet any unattached
cells. The cell-free media were immediately transferred to a centrifuge
tube containing an equal volume of ice-cold 10% (w/v) perchloric acid,
0.2 M boric acid, and 20 [micro]M [gamma]-glutamyl glutamate as an
internal standard [33]. Extracts were centrifuged at 16,000 xg for 2
minutes to remove precipitated protein. The protein-free extracts were
derivatized with iodoacetic acid and dansyl chloride and analyzed by
HPLC (Waters Corporation, Millford, MA) as previously described [34].
Concentrations of thiols and disulfides were determined by integration
relative to the internal standard. Redox potentials ([E.sub.h]) of the
GSH/GSSG and Cys/CySS pools, given in millivolts (mV), were calculated
from concentrations of GSH, GSSG, and Cys and CySS in molar units with
the following forms of the Nernst equation for pH 7.4: [E.sub.h]
GSH/GSSG = -264+30 *log([GSSG]/[[GSH].sup.2]); [E.sub.h] Cys/CySS = -250
+ 30 * log([CySS]/[[Cys].sup.2]) [26].

2.6. Microarray Analysis. Biotinylated cRNA was prepared from total
RNA using Affymetrix GeneChip Whole Transcript (WT) Plus Reagent kit.
Fragmented cRNA was hybridized to Affymetrix MoGene-2_0-st-v1 arrays and
processed on an Affymetrix FS-450 fluidics station and scanned on an
Affymetrix GeneChip scanner. The resulting .cel files were imported into
Partek Genomics Suite 6.6 (6.15.0327) and transcripts were normalized
and summarized using RMA default settings. Samples were compared using a
one-way ANOVA model to assess the contribution of age. A step-up false
discovery rate was applied as multiple test correction for the resulting
p values.

2.8. Statistical Analysis. All data are expressed as mean [+ or -]
standard deviation. Unpaired two-tailed E-tests and one-way analysis of
variance tests were used for single and multiple comparisons,
respectively (p values < 0.05 were considered significant). Posttest
analysis was performed using Dunnett's multiple comparison test to
compare between groups. GraphPad Prism and GraphPad In-Stat version 4
software (GraphPad Software, Inc., La Jolla, CA) were used to calculate
the statistics.

3. Results

3.1. Extracellular [E.sub.h]CySS Is Regulated Differently by Lung
Fibroblasts from Young and Old Mice. Fibroblasts isolated from the lungs
of a young mouse conditioned the culture medium to achieve an
extracellular [E.sub.h]CySS of about -95 mV within 24 hours, independent
of the starting redox potential of the medium (Figure 1(a)). In
contrast, cells from old mice challenged with oxidizing media (0 mV) or
with physiological redox media (-80 mV) stabilized at a redox potential
that was about 40 mV more oxidizing than that achieved by young cells
(Figure 1(b)). When old cells were challenged with a reducing
environment (-150 mV), they did not quite return to the same level as
when cultured in either oxidizing (0 mV) or typical (-80 mV) media but
were still more oxidizing than was seen in the young cell cultures.

In the absence of cells, all 3 redox media became more oxidized
over the 24-hour incubation period (Figure 1(c)). CySS concentrations
were quite stable in all 3 media over 24 hours, but Cys concentrations
dropped dramatically. Thus, oxidation of the media redox potential in
the absence of cells reflected a decrease in Cys rather than an increase
in CySS (Figures 2(a) and 2(b)). The pattern of changes in CySS and Cys
was different in the presence of cells. In the presence of young cells,
CySS concentrations in the conditioned media were lower than in the
presence of old cells (Figures 2(c) and 2(d)). CySS concentrations were
decreased by 60 to 75% over 24 hours in the young cell culture media,
whereas there was only a 30% decrease in CySS in the old cell media.

Some of the CySS in both young and old cultures appeared to be
metabolized to Cys. Cys concentrations in conditioned media from both
young and old cell cultures, and in all 3 redox media, were higher than
the Cys concentrations seen in the corresponding redox media incubated
in the absence of cells (compare Figures 2(c) and 2(d) to 2(a) and
2(b)). Both young and old cells produced Cys at rates that exceeded the
spontaneous degradation observed in the absence of cells. However, old
cells reached a lower steady state level of Cys than young cells.

3.2. Rate of CySS Consumption Is Slower in Old Cells Than in Young
Cells. The data in Figure 2 suggested that young cells removed CySS from
the media more rapidly than old cells. To investigate this further, we
measured CySS concentrations in media to which CySS was added as a
function of incubation time. Although CySS did not change in the absence
of cells, there was an exponential decline in extracellular CySS in both
the young and old cell cultures (Figure 3(a)). The data were plotted as
the natural log of the CySS concentration, and the slope of the line was
used to calculate the half-life of CySS in each culture. Accordingly,
media on young cells exhibited a CySS half-life of 10.5 hours, whereas
media on old cells had a CySS half-life of 17.0 hours. In contrast, the
half-life of Cys in media was 5 hours, and the presence of either young
or old cells had no effect on the stability of Cys (Figure 3(b)).

3.3. The Rate of Equilibration of the Extracellular Redox
Environment Is Slower in Old Cells Than in Young Cells. The data in
Figure 1 showed that redox equilibrium is restored within 24 hours after
it is perturbed. Here, we examined earlier time points to determine how
quickly equilibrium is reestablished and whether there is a difference
between old and young cells. When challenged with a reducing environment
(-150 mV), both young and old cells moved toward their preferred
[E.sub.h]CySS set point at equal rates (Figure 4). In contrast, the
response of young and old cells to an oxidizing environment was
different: by 1 hour, young cells had nearly reached their set point
while old cells were only just beginning to reduce the medium. By 4
hours, however, both young and old cells had restored their media to 95
mV and -60 mV, respectively. When compared with Figure 1, it can be seen
that these respective [E.sub.h]CySS values were maintained for at least
24 hours.

3.4. The CySS Transporter Is Downregulated in Old Fibroblasts. The
abovementioned data suggest that old fibroblasts may be deficient in
cystine transport. To begin to assess this, we examined the expression
of the CySS transporter, specifically the Slc7a11 subunit of the xCT
transporter that confers specificity [36]. As predicted from the
observed decreased rate of CySS uptake, there was 10-fold lower
expression of Slc7a11 in lung fibroblasts harvested from aged animals
when compared to those obtained from young animals (Figure 5(a)).

3.5. Aging Is Not Associated with Global Changes in Expression of
Antioxidant and Thiol-Regulating Enzymes. To further assess changes that
occurred with aging, microarray analysis was used to identify genes that
were differentially expressed in fibroblasts as a function of age. Table
1 lists genes that could contribute to differences in the extracellular
redox states through their roles as Cys and CySS transporters and
glutathione-related thiol-disulfide regulating enzymes. Table 2 lists
thioredoxin-related thiol-disulfide regulating enzymes and antioxidant
defense enzymes. Remarkably, only 2 of these genes exhibited greater
than 2-fold difference between young and old cells: Slc7a11 and Sod3. As
discussed above, Slc7a11 encodes the CySS- and Glu-specific subunit of
the xCT transport system. Sod3 encodes the extracellular superoxide
dismutase that catalyzes the dismutation of superoxide anion to hydrogen
peroxide and molecular oxygen. If the stringency of the comparison is
lowered by removing the fold-change cut-off and relying solely on the
adjusted p value to assign significance, 6 more genes are added to the
list. Of these, Slc1a4 and Glx are downregulated in aging fibroblasts,
whereas the other 4 are upregulated, and, therefore, would not explain
the increase in extracellular redox potential.

3.6. Old Fibroblasts Express Higher Levels of Fibronectin EDA and
Other Components of the Extracellular Matrix. Like aging, culturing
fibroblasts in oxidizing media promote their transdifferentiation into
myofibroblasts, characterized by increased expression of fibronectin
(Fn1), transforming growth factor-[beta] (Tgfb1), and a-smooth muscle
actin (Acta2) [30, 37]. Because old fibroblasts naturally condition
their media to a more oxidizing state than young fibroblasts (see Figure
1(a)), we reasoned that these cells would express higher levels of Fn1
and its profibrotic splice variant FnEDA. Indeed, old fibroblasts had
18-fold higher levels of fibronectin EDA mRNA than young fibroblasts
(Figure 5(b)). Examination of the microarray data confirmed that aging
fibroblasts had increased expression of Fn1, Tgfb1, and Acta2 (Table 3).
In addition, 4 different collagen genes and Tgfb3 were also upregulated
(Table 3). Two laminins that have been reported to be downregulated in
the aging lung, Lama3 and Lama4 [38], were downregulated in the
fibroblasts from old mice. A third laminin, Lama5, was upregulated in
fibroblasts from old mice (Table 3). Thus, production of extracellular
matrix components can be affected by either artificial manipulation of
the extracellular [E.sub.h]CySS [30] or age-related differences in the
set point of the extracellular [E.sub.h]CySS.

4. Discussion

In the current study, we found that primary lung fibroblasts from
young mice condition their media close to -95 mV within 4 hours of a
media change. This value is within the range of -90 to -100 mV reported
for mouse plasma [E.sub.h]CySS [39-41] but is more reduced than the
average value of -80 mV observed in human plasma [24, 26, 42-44]. These
data support the concept that mice and humans have different optimal
plasma [E.sub.h]CySS set points and that cells derived from these
species reproduce these set points when placed in culture. When human
cancer cell lines HT-29 and Caco2 are grown in culture, they condition
their extracellular [E.sub.h]CySS to -80 mV, and stimulation with growth
factors increases the rate at which they achieve homeostasis [27, 45,
46]. In a study of murine bone marrow derived dendritic cells, Yan et
al. found that the cells reached an extracellular [E.sub.h]CySS of-110
mV, but only when stimulated to proliferate by coculturing with T cells
[29]. The primary mouse fibroblasts used in the present study were
actively proliferating and rapidly achieved an extracellular redox
potential that was consistent with those observed in mice and mouse cell
cultures, but that was more reducing than those observed in humans and
human cell cultures.

Plasma redox potentials become more oxidizing with age, but the
reasons for this are unclear. We now report that cells isolated from an
old mouse retain an apparent preference for an oxidizing extracellular
environment or are unable to create an optimally reducing environment.
We found that fibroblasts from old mice expressed much lower levels of
the CySS-specific transporter subunit Slc7a11 than fibroblasts from
young mice. This finding was consistent with a decreased rate of CySS
removal from the media and a decreased rate of reduction of an oxidizing
environment. To metabolize extracellular CySS to Cys, the cells must
import CySS, reduce it to Cys [47], then export Cys back into the
extracellular space. At least 2 enzymes have been shown to have
CySS-reducing activity: thioredoxin 1 (Txn1) and thioredoxin domain
containing 17 (Txndc17), both of which receive electrons from
thioredoxin reductase (Txnrd1) [47]. However, expression of the genes
encoding these enzymes was unchanged in aging fibroblasts. Therefore,
our data support the interpretation that import of CySS, rather than
reduction of CySS to Cys, is impaired in aging cells. However, it will
be important to determine whether the activities, not just mRNA levels,
of these CySS reductases remain unchanged in aging fibroblasts.

The expression of extracellular Sod3 was also lower in old
fibroblasts than in young fibroblasts. Loss of this important
antioxidant from the extracellular compartment could contribute to the
shift toward more oxidizing conditions that we observed in this study.
The slower rate of CySS decline from the conditioned media of old
fibroblasts relative to young fibroblasts could be the result of either
a slower rate of cellular uptake (by Slc7a11) or an increased rate of
its formation via oxidation of Cys. Cu,Zn-SOD (Sod1) can catalyze the
oxidation of Cys in the presence of oxygen [48], but the decrease in
expression of extracellular Cu,Zn-SOD (Sod3) that we observed does not
support this mechanism in aging. It is possible that the amount of SOD
protein or SOD activity is increased in the extracellular compartment of
aging fibroblasts, despite the observed decrease in expression. Future
studies will need to investigate this potential mechanism.
Alternatively, increased extracellular superoxide concentrations as a
result of lower extracellular Cu,Zn-SOD activity could lead to increased
rates of Cys oxidation via direct or indirect reactions [49]. However,
we did not observe a corresponding increase in the rate of Cys
disappearance from the media, supporting the conclusion that differences
in CySS uptake are one of the major forces leading to changes in steady
state CySS concentrations between young and old cells.

While our results point to decreased expression of the CySS
transporter as one of the major factors leading to oxidation of the
extracellular redox state of aging cells, there are other potential
mechanisms that could be contributing. For example, mitochondrial
production of superoxide, hydrogen peroxide, and other oxidizing species
tends to increase with age [50]. Increased oxidant production within the
mitochondria can lead to oxidation of cytoplasmic targets [51], but it
is unclear what effect this would have on extracellular redox state. An
increased oxidant burden in the cytoplasmic compartment could decrease
the rate of CySS reduction to Cys, consistent with results of the
present study showing that extracellular Cys concentrations are lower in
cultures of fibroblasts from old mice. Further studies will be needed to
assess the relative contributions of decreased import of extracellular
CySS and decreased reduction of intracellular CySS in regulating the
balance between CySS and Cys in the extracellular space.

Cells not only regulate their extracellular environment but also
respond to it. In the present study, we incubated cells in media with
[E.sub.h]CySS values ranging from 0 mV to -150 mV to investigate how
well cells restored the [E.sub.h]CySS to their preferred set point. We
and others have used a similar approach to demonstrate that incubating
cells in different redox media affects proliferation [46], cell adhesion
[52], inflammatory signaling [53-55], cancer cell invasiveness [56, 57],
membrane receptor activation [58], and expression of extracellular
matrix proteins [30]. For example, adding a 0 mV medium to cells results
in higher expression of genes indicative of myofibroblast
differentiation than adding -150 mV medium. The present study shows that
cells from an old mouse produce an extracellular redox environment that
mimics this effect of artificially adding an oxidizing environment.
Therefore, cells are not just passively responding to changes in
extracellular [E.sub.h]CySS; they are changing the extracellular
[E.sub.h]CySS. Interestingly, the cells become more myofibroblast-like
in an oxidized extracellular environment regardless of whether the
oxidation arises as a result of external forces or by its own
manipulation of the environment.

5. Conclusions

Fibroblasts play a key role in remodeling the extracellular matrix
in response to stress, but it is unknown how they contribute to
extracellular redox remodeling. Here, we show that fibroblasts play an
active role in controlling their extracellular redoxenvironment and that
fibroblasts from old mice are either unable to attain an ideal
[E.sub.h]CySS or have been reprogrammed to maintain a more oxidizing set
point. These results may explain the subtle profibrotic remodeling of
the extracellular matrix that occurs with aging and lead to a better
understanding of how changing extracellular redox potential increases
susceptibility to lung injury.

http://dx.doi.org/10.1155/2016/1561305

Disclosure

The content is solely the responsibility of the authors and does
not necessarily represent the official views of the National Institutes
of Health.

Competing Interests

The authors declare that they have no competing interests.

Acknowledgments

Research reported in this publication was supported by Veterans
Affairs Grant 5I01 BX000216-02 (Roman) and by R01 AA019953 (Roman), U01
HL121807 (Roman), R56HL128597 (Zelko), the National Institute on Alcohol
Abuse and Alcoholism under Award no. P50AA024337-8305 (Roman), and an
Institutional Development Award (IDeA) from the National Institute of
General Medical Sciences of the National Institutes of Health under
Grant no. P20GM113226-6176 (Watson).

(1) Department of Medicine, Division of Gastroenterology,
Hepatology and Nutrition, University of Louisville School of Medicine,
Louisville, KY, USA

(2) Department of Pharmacology and Toxicology, University of
Louisville School of Medicine, Louisville, KY, USA

(3) Department of Biochemistry and Molecular Genetics, University
of Louisville School of Medicine, Louisville, KY, USA

(4) Department of Medicine, Division of Pulmonary, Critical Care,
and Sleep Medicine, University of Louisville School of Medicine,
Louisville, KY, USA

(5) Robley Rex Veterans Affairs Medical Center, Louisville, KY, USA

Correspondence should be addressed to Walter H. Watson;
bert.watson@louisville.edu

Received 5 May 2016; Accepted 7 August 2016

Academic Editor: Jean-Claude Lavoie

Caption: Figure 1: [E.sub.h]CySS in media conditioned by
fibroblasts isolated from the lungs of young and old mice. Primary lung
fibroblasts from young mice and old mice were cultured in one of 3
different redox media: oxidizing (0 mV), typical (-80 mV), or reducing
(-150 mV). Media was removed immediately (0 hours) or after 24 hours and
processed for determination of Cys and CySS concentrations and
[E.sub.h]CySS as described in Section 2. (a) Change in [E.sub.h]CySS of
3 different redox media following 24-hour incubation with cells from
young mice. (b) Change in [E.sub.h]CySS of 3 different redox media
following 24-hour incubation with cells from old mice. (c) Change in
[E.sub.h]CySS of 3 different redox media following 24-hour incubation in
the absence of cells.

Caption: Figure 2: Cys and CySS concentrations in media conditioned
by fibroblasts isolated from the lungs of young and old mice. (a) Cys
and CySS concentrations in redox media at time 0 hours and (b) after 24
hours of incubation at 37[degrees]C in the absence of cells. (c) Cys and
CySS concentrations in the conditioned media of fibroblasts from young
and (d) old mice after 24 hours of incubation at 37[degrees]C.

Caption: Figure 3: Extracellular CySS is consumed more rapidly by
fibroblasts from young mice than by fibroblasts from old mice. DMEM
containing 45 [micro]M CySS (a) or 100 [micro]M Cys (b) was added to
lung fibroblasts from young and old mice. Media were removed at
different time points for analysis of Cys and CySS concentrations by
HPLC. Concentrations were transformed as the natural logarithm. The line
through the data represents the best fit via linear regression.

Caption: Figure 4: Time course of [E.sub.h]CySS restoration by
young and old fibroblasts after challenge with oxidizing or reducing
media. Lung fibroblasts from young and old mice were incubated with
either 0 mV media or -150 mV media for 0, 1, or 4 hours. Conditioned
media were removed and analyzed for Cys and CySS concentrations by HPLC.

Caption: Figure 5: Expression levels of the CySS transporter
subunit Slc7a11 and the fibronectin EDA splice variant in young and old
fibroblasts. Lung fibroblasts from young and old mice were plated in
6-well plates at a density of 800,000 cells per well in complete DMEM.
Cells were collected 24 hours later, and RNA was isolated for RT-PCR
analysis. (a) Decreased expression of Slc7a11 in lung fibroblasts
harvested from aged animals when compared to those obtained from young
animals. (b) Increased expression of fibronectin EDA in lung fibroblasts
harvested from aged animals when compared to those obtained from young
animals (* P < 0.001).

Table 1: Differential expression of genes related to Cys and CySS
transporters and glutathione-related thiol-disulfide enzymes.
Old versus
young (fold- Step-up p
Gene difference) (a) value (b)
Cys and CySS
transporters
Slc7all# Down 5.0-fold# 0.0034#
Slc7a9 0.28
Slc1a1 0.85
Slc1a4 Down 1.5-fold 0.034#
Slc1a5 0.092
Slc3a1 0.38
Slc3a2 0.69
Thiol-disulfide
enzymes:
Glutathione-related
Gpx1 Up 1.4-fold 0.033#
Gpx2 0.096
Gpx3 0.49
Gpx4 0.064
Gpx5 0.87
Gpx6 0.71
Gpx7 0.067
Gpx8 0.23
Gsr 0.54
Gclc 0.62
Gclm 0.60
Gss 0.48
Glrx Down 1.7-fold 0.0097#
Glrx2 0.11
Glrx3 Up 1.4-fold 0.044#
Glrx5 Up 1.2-fold 0.048#
(a) Fold-difference in expression in old fibroblasts relative
to the expression in young fibroblasts is shown for all genes
with a significant difference. Those exceeding the cut-off
threshold of 2-fold are shown in bold.
(b) Step-up false discovery rate for the comparison between
young and old cells following correction for multiple comparisons.
Note: Those exceeding the cut-off
threshold of 2-fold are indicated with #.
Table 2: Differential expression of genes related to
thioredoxin-related thiol-disulfide enzymes and antioxidant
enzymes.
Old versus
young (fold- Step-up p
Gene difference) (a) value (b)
Thiol-disulfide
enzymes: thioredoxin-
related
Prxd1 0.62
Prdx2 0.35
Prdx3 Up 1.4-fold 0.0074#
Prdx4 0.20
Prdx5 0.29
Prdx6 0.40
Prdx6b 0.93
Srxn1 0.64
Txn1 0.77
Txn2 0.79
Txnrd1 0.65
Txnrd2 0.91
Txnrd3 0.25
Txndc17 0.24
Txnip 0.09
Antioxidant
enzymes
Sod1 0.32
Sod2 0.17
Sod3 Down 4.l-fold# 0.0074#
Cat 0.75
G6pd2 0.99
(a) Fold-difference in expression in old fibroblasts relative
to the expression in young fibroblasts is shown for all genes
with a significant difference. Those exceeding the cut-off
threshold of 2-fold are shown in bold.
(b) Step-up false discovery rate for the comparison between
young and old cells following correction for multiple comparisons.
Note: Those exceeding the cut-off
threshold of 2-fold are indicated with #.
Table 3: Differential expression of genes related to the extracellular
matrix in primary lung fibroblasts from young and old mice. Only
genes that were significantly different between old and young cells
are shown.
Old versus
young (fold- Step-up p
Gene difference) (a) value (b)
Myofibroblast
transdifferentiation
Tgfb1 Up 1.6-fold 0.038#
Tgfb3# Up 6.0-fold# 0.0033#
Acta2# Up 2.2-fold# 0.022#
Fn1 Up 1.3-fold 0.0091#
Col4a1# Up 3.2-fold# 0.016#
Col4a2# Up 2.3-fold# 0.0096#
Col5a2 Up 1.3-fold 0.048#
Col11a1# Up 3.5-fold# 0.0033#
Lama3 Down 1.3-fold 0.023#
Lama4# Down 16.4-fold# 0.0016#
Lama5# Up 6.0-fold# 0.0014#
(a) Fold-difference in expression in old fibroblasts relative to
the expression in young fibroblasts is shown for all genes with
a significant difference. Those exceeding the cut-off threshold
of 2-fold are shown in bold.
(b) Step-up false discovery rate for the comparison between young
and old cells following correction for multiple comparisons.
Note: Those exceeding the cut-off threshold
of 2-fold are indicated with #

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